The use of genetic transformation technology makes it possible to impart new traits to plants by expressing a useful gene in a target plant. A wide range of genetically modified plants produced in this manner have already been cultivated. Since regulation of gene expression is mainly controlled at the level of transcription, transcriptional regulation is the most important in terms of regulating the expression of genes. Namely, expressing a gene at a suitable time, in a suitable tissue and at a suitable strength is important for producing an industrially useful genetically modified plant. In many cases, transcription is control by a DNA sequence on the 5′ untranslated region of a open reading frame. A region of DNA that determines the starting site of gene transcription and directly regulates the frequency thereof is referred to as a promoter. A promoter is located in a start codon consisting of several tens of base pairs (bp) on the 5′-untranslated region, and frequently contains a TATA box and the like. A cis element that binds various transcriptional regulatory factors is also present on the 5′-untranslated region, and the presence thereof serves to control the timing of transcription, the tissue in which transcription takes place and transcriptional strength. Transcriptional regulatory factors are classified into many families according to their amino acid sequence. For example, examples of well-known families of transcriptional regulatory factors include Myb transcriptional regulatory factor and bHLH (basic helix loop helix) regulatory factor. In actuality, the terms transcriptional regulatory factor and promoter are frequently used with the same meaning.
Anthocyanins, which compose the main components of flower color, are a member of secondary metabolites generically referred to as flavonoids. The color of anthocyanins is dependent on their color. Namely, the color becomes blue as the number of hydroxyl groups of the B ring of anthocyanidins, which is the chromophore of anthocyanins, increases. In addition, as the number of aromatic acyl groups (such as coumaroyl groups or caffeolyl groups) that modify the anthocyanin increases (namely, the wavelength of maximum absorbance shifts to a longer wavelength), the color of the anthocyanin becomes blue and the stability of the anthocyanin is known to increase (see Non-Patent Document 1).
Considerable research has been conducted on those enzymes and genes that encode those enzymes involved in the biosynthesis of anthocyanins (see, Non-Patent Document 1). For example, an enzyme gene that catalyzes a reaction by which an aromatic acyl group is transferred to anthocyanin is obtained from Japanese gentian, lavender and petunias (see Patent Document 1 and Patent Document 2). An enzyme gene involved in the synthesis of anthocyanin that accumulates in the leaves of red perilla (malonylshisonin, 3-0-(6-0-(E)-p-coumaroyl-β-D-glucopyranosyl)-5-0-(6-0-malonyl-β-D-glucopyranosyl)-cyanidin) (see Non-Patent Document 2) has previously been reported in hydroxycinnamoyl CoA: anthocyanin-3-glucoside-aromatic acyl group transferase (3AT) gene (or more simply referred to as “shiso (perilla) anthocyanin-3-acyltransferase (3AT) gene”) (see Patent Document 1). Moreover, findings have also been obtained regarding the transcriptional regulation (control) of biosynthase genes of anthocyanins. Cis element sequences bound by Myb transcriptional regulatory factor and bHLH transcriptional regulatory factor are present in the transcriptional regulatory region located on the 5′-region of the start codons of these genes. Myb transcriptional regulatory factor and bHLH transcriptional regulatory factor are known to control synthesis of anthocyanins in petunias, corn and perilla (see Non-Patent Document 1).
Promoters (also referred to as transcriptional regulatory regions) responsible for gene transcription in plants consist of so-called constitutive promoters, which function in any tissue and at any time such as in the developmental stage, organ/tissue-specific promoters, which only function in specific organs and tissues, and time-specific promoters, which only express at a specific time of the developmental stage. Constitutive promoters are frequently used as promoters for expressing useful genes in genetically modified plants. Typical examples of constitutive promoters include cauliflower mosaic virus 35S promoter (also abbreviated as CaMV35S promoter) and promoters construction on the basis thereof (see Non-Patent Document 3), and Mac1 promoter (see Non-Patent Document 4). In plants, however, many genes are only expressed in specific tissues or organs or are expressed time-specifically. This suggests that tissue/organ-specific or time-specific expression of genes is necessary for plants. There are examples of genetic recombination of plants that utilize such tissue/organ-specific or time-specific transcriptional regulatory regions. For example, there are examples of protein being accumulated in seeds by using a seed-specific transcriptional regulatory region.
However, although plants produce flowers of various colors, there are few species capable of producing flowers of all colors due to genetic restrictions on that species. For example, there are no varieties of rose or carnation in nature that are capable of producing blue or purple flowers. This is because roses and carnations lack the flavonoid 3′,5′-hydroxylase gene required to synthesize the anthocyanidin, delphinidin, which is synthesized by many species that produce blue and purple flowers. By transformation with the flavonoid 3′,5′-hydroxylase gene of petunia or pansy, for example, which are specifies capable of producing blue and purple flowers, into these species, these species can be made to produce blue flowers. In the case of carnations, the transcriptional regulatory region of chalcone synthase gene derived from common snapdragon or petunia is used to transcribe flavonid 3′,5′-hydroxylase gene derived from common snapdragon or petunia. Examples of plasmids containing the transcriptional regulatory region of chalcone synthase gene derived from common snapdragon or petunia include plasmids pCGP485 and pCGP653 described in Patent Document 3, and examples of plasmids containing a constitutive transcriptional regulatory region include plasmid PCGP628 (containing a Mac1 promoter) and plasmid pSPB130 (containing a CaMV35S promoter to which is added E12 enhancer) described in Patent Document 4.
However, it is difficult to predict how strongly such promoters function in recombinant plants to be able to bring about a target phenotype. In addition, since repeatedly using the same promoter to express a plurality of foreign genes may cause gene silencing, it is thought that this should be avoided (see Non-Patent Document 5).
Thus, although several promoters have been used to change flower color, a useful promoter corresponding to the host plant and the objective is needed in order to further change to a different flower color.
In particular, chrysanthemum plants (also simply referred to as chrysanthemums) account for about 30% of all wholesale flower sales throughout Japan (Summary of 2007 Flowering Plant Wholesale Market Survey Results, Ministry of Agriculture, Forestry and Fisheries), making these plants an important product when compared with roses accounting for roughly 9% and carnations accounting for roughly 7%. Although chrysanthemums come in flower colors including white, yellow, orange, red, pink and purplish red, there are no existing varieties or closely related wild varieties that produce bluish flowers such as those having a purple or blue color.
Thus, one objective of the selective breeding of bluish flowers is to stimulate new demand. Chrysanthemum flower color is expressed due to a combination of anthocyanins and carotenoids. Anthocyanins are able to express various colors due to differences in the structure of the anthocyanidin serving as the basic backbone, and differences in modification by sugars and organic acids. However, there are known to be two types of anthocyanins that govern chrysanthemum flower color in which cyanidin at position 3 is modified by glucose and malonic acid (cyanidin 3-0-(6″-0-monomalonyl-β-glucopyranoside and 3-0-(3″,6″-0-dimalonyl-β-glucopyranoside) (see Non-Patent Document 6). In addition, these structures are comparatively simple (see FIG. 1). This causes the range of flower color attributable to anthocyanins in chrysanthemums to be extremely narrow. However, although the expression of bluish color is primarily the result of anthocyanins, since there is no gene that encodes the key enzyme of flavonoid 3′,5′-hydroxylase (F3′5′H) in chrysanthemums, delphinidin-based anthocyanin, which produces blue color, is not biosynthesized in chrysanthemums (see FIG. 1). Therefore, the development of a technology has been sought for controlling the expression of chrysanthemum anthocyanins using genetic engineering techniques in order to be able to produce a chrysanthemum that produces bluish flowers by modifying anthocyanin-based pigment that accumulates in chrysanthemum petals.
As was previously described, although chrysanthemums are the most important flowering plant in Japan, since they are hexaploidal resulting in high ploidy and have a large genome size, in addition to having low transformation efficiency, since they may also cause silencing (deactivation) of transgenes, it is not easy to obtain genetically modified chrysanthemums capable of stable transgene expression. In chrysanthemums transformed with β-glucuronidase (GUS) gene coupled to CaMV35S promoter, the activity of the GUS gene is roughly one-tenth that of tobacco transformed with the same gene, and that activity has been reported to decrease in nearly all individuals after 12 months have elapsed following transformation (see Non-Patent Document 7). Although a promoter of a chlorophyll a/b-bound protein that favorably functions in chrysanthemums has been reported to have been obtained in order to stably express an exogenous gene in chrysanthemums, this promoter is not suitable for expressing genes in flower petals in which there is little chlorophyll present (see Non-Patent Document 8). In addition, when GUS gene coupled to tobacco elongation factor 1 (EF1α) promoter is transformed into chrysanthemums, GUS gene has been reported to be expressed in leaves and petals even after the passage of 20 months or more (see Non-Patent Document 9). Moreover, there are also examples of flower life being prolonged by expressing a mutant ethylene receptor gene in chrysanthemums (see Non-Patent Document 10), flower form being changed by suppressing expression of chrysanthemum AGAMOUS gene (see Non-Patent Document 11), and expression of exogenous genes being increased in chrysanthemums (see Non-Patent Document 12) by using a translation enhancer of tobacco alcohol dehydrogenase (see Patent Document 7).
On the other hand, although there have been examples of successful alteration of chrysanthemum flower color by genetic recombination, including a report of having changed pink flowers to white flowers by suppressing the chalcone synthase (CHS) gene by co-suppression (see Non-Patent Document 13), and a report of having changed white flowers to yellow flowers by suppressing carotenoid cleavage dioxygenase (CCD4a) by RNAi (see Non-Patent Document 14), all of these methods involve alteration of flower color by suppressing expression of endogenous genes, and there have been no successful examples of altering flower color by over-expression of exogenous genes as well as no examples of having realized a change in anthocyanin structure or an accompanying change in flower color.
Although attempts to alter flower color by over-expression of an exogenous gene have been reported that involve transformation with a gene encoding F3′5′H, which is an enzyme required for synthesis of delphinidin (see Patent Document 5 and Non-Patent Document 15), the delphinidin produced due to the action of the transfected F3′5′H gene accumulates in ray petals, and there are no reports of the production of bluish chrysanthemums. In chrysanthemums, even if F3′5′H is expressed with CaMV35S promoter, production of delphinidin is not observed (see Non-Patent Document 15). In addition, expression of a gene expressed with CaMV35S promoter is unsuitable for stable expression, and for example, ends up dissipating accompanying growth of the chrysanthemum transformant (see Non-Patent Document 7). Potato Lhca3.St.1 promoter (see Non-Patent Document 16), chrysanthemum UEP1 promoter (see Non-Patent Document 17) and tobacco EF1α promoter (see Patent Document 6 and Non-Patent Document 9), for example, have been developed for use as promoters enabling efficient and stable expression of exogenous genes in the ray petals of chrysanthemums. However, there have been no reports describing alteration of chrysanthemum flower color by over-expression of an exogenous gene using these promoters. On the basis of the above, in order to produce chrysanthemums in which flower color has been altered by genetic recombination, it is necessary to establish a technology for controlling the expression of flavonoid biosynthesis genes, including the development of a promoter suitable for chrysanthemums.
Although gene expression is mainly controlled by transcriptional regulatory regions, sequences are also known that improve translation of mRNA. For example, the omega sequence derived from tobacco mosaic virus is known to increase the translation efficiency of heterologous genes coupled to the omega sequence both in vitro and in vivo (see Non-Patent Document 18). In addition, a sequence (ADH200) present in the 5′-untranslated region of tobacco alcohol dehydrogenase (NtADH5′UTR) is known to contribute to improved stability of the expression of heterologous genes (see Patent Document 7). In addition, in the case of coupling a 94 bp translation enhancer (ADHNF, see Patent Document 8) present downstream from this sequence to the 3′-side of CaMV35S promoter and further transformation with an expression cassette coupled with GUS gene, this sequence has been reported to contribute to increased translation efficiency in chrysanthemums (see Non-Patent Document 12). However, there are no examples of this sequence being used to change flower color by altering the structure and composition of flavonoids. Since it is necessary to express a heterologous gene in epidermal cells in which flavonoids and anthocyanins primarily accumulate in order to alter flower color, it is difficult to infer from conventional results whether or not NtADH5′UTR (ADH200 or translation enhancer ADHNF) is effective for altering flower color.